RNA in all cellular organisms is synthesized by a complex molecular machine, the DNA-dependent RNA polymerase (RNAP). In its simplest bacterial form, the enzyme comprises at least 4 subunits with a total molecular mass of around 400 kDa. The eukaryotic enzymes comprise upwards of a dozen subunits with a total molecular mass of around 500 kDa. The essential core component of the RNAP (subunit composition .alpha..sub.2.beta..beta.') is evolutionarily conserved from bacteria to man [Archambault and Friesen, Microbiological Reviews, 57:703-724 (1993)]. Sequence homologies point to structural and functional homologies, making the simpler bacterial RNAPs excellent model systems for understanding the multisubunit cellular RNAPs in general.
The basic elements of the transcription cycle were elucidated through study of the prokaryotic system. In this cycle, the RNAP, along with other factors, locates specific sequences called promoters within the double-stranded DNA, forms the open complex by melting a portion of the DNA surrounding the transcription start site, initiates the synthesis of an RNA chain, and elongates the RNA chain completely processively while translocating itself and the melted transcription bubble along the DNA template. Finally it releases itself and the completed transcript from the DNA when a specific termination signal is encountered. The current view is that the transcribing RNAP contains sites for binding the DNA template as well as forming and maintaining the transcription bubble, binding the RNA transcript, and binding the incoming nucleotide-triphosphate substrate.
From the initial indications of DNA-dependent RNAP activity from a number of systems, [Weiss and Gladstone, J. Am. Chem. Soc., 81:4118-4119 (1959)]; Hurwitz et al., Biochem. Biophys. Res. Commun., 3:15 (1960); Stevens, Biochem. Biophys. Res. Commun., 3:92 (1960); Huang et al., Biochem. Biophys. Res. Commun., 3:689 (1960); and Weiss and Nakamoto, J. Biol. Chem., 236:PC 19 (1961)], and the isolation of the RNAP enzyme from bacterial sources [Chamberlin and Berg, Proc. Natl. Acad. Sci. USA, 48:81-94 (1962)], a wealth of biochemical, biophysical, and genetic information has accumulated on RNAP and its complexes with nucleic acids and accessory factors. Nevertheless, the enzyme itself, in terms of its structure/function relationship, remains a black box. An essential step towards understanding the mechanism of transcription and its regulation is to determine three-dimensional structures of RNAP and its complexes with DNA, RNA, and regulatory factors [von Hippel et al., Annual reviews of Biochemistry, 53:389-446 (1984); Erie et al., Annual Review of Biophysics & Biomolecular Structure, 21:379-415 (1992); Sentenac et al., Transcriptional Regulation (eds. McKnight, S. L. & Yamamoto, K. R.) 27-54 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1992); Gross et al., Philosophical Transactions of the Royal Society of London--Series B:Biological Sciences, 351:475-482 (1996); and Nudler, J. Mol. Biol., 288:1-12 (1999)].
The key feature of low-resolution structures of bacterial and eukaryotic RNAPs, provided by electron crystallography, is a thumblike projection surrounding a groove or channel that is an appropriate size for accommodating double-helical DNA [Darst et al., Nature, 340:730-732 (1989); Darst et al., Cell, 66:121-128 (1991); Schultz et al., EMBO J., 12:2601-2607 (1993); Polyakov et al., Cell, 83:365-373 (1995); Darst et al., J. Structural Biol., 124:115-122 (1998); and Darst et al., Cold Spring Harbor Symp. Quant. Biol., 63:269-276 (1998)].
Bacterial infections remain among the most common and deadly causes of human disease. Infectious diseases are the third leading cause of death in the United States and the leading cause of death worldwide [Binder et al., Science 284:1311-1313 (1999)].
Although, there was initial optimism in the middle of this century that diseases caused by bacteria would be quickly eradicated, it has become evident that the so-called "miracle drugs" are not sufficient to accomplish this task. Indeed, antibiotic resistant pathogenic strains of bacteria have become common-place, and bacterial resistance to the new variations of these drugs appears to be outpacing the ability of scientists to develop effective chemical analogs of the existing drugs [See, Stuart B. Levy, The Challenge of Antibiotic Resistance, in Scientific American, 46-53 (March, 1998)]. Therefore, new approaches to drug development are necessary to combat the ever-increasing number of antibiotic-resistant pathogens.
Classical penicillin-type antibiotics effect a single class of proteins known as autolysins. Thus, the development of new drugs which effect an alternative bacterial target protein would be desirable. Such a target protein ideally would be indispensable for bacterial survival. A enzyme such as bacterial RNAP would thus be a prime candidate for such drug development.
Therefore, there is a need to develop methods for identifying drugs that interfere with bacterial RNAP. Unfortunately, such identification has heretofore relied on serendipity and/or systematic screening of large numbers of natural and synthetic compounds. One superior method for drug screening relies on structure based rational drug design. In such cases, a three dimensional structure of the protein or peptide is determined and potential agonists and/or antagonists are designed with the aid of computer modeling [Bugg et al., Scientific American, Dec.: 92-98 (1993); West et al., TIPS, 16:67-74 (1995); Dunbrack et al., Folding & Design, 2:27-42 (1997)].
Therefore, there is a need for obtaining a crystal of the bacterial RNAP that is amenable to high resolution X-ray crystallographic analysis. In addition, there is a need for determining the three-dimensional structure of the RNAP. Furthermore, there is a need for developing procedures of structure based rational drug design using such three-dimensional information. Finally, there is a need to employ such procedures to develop new anti-bacterial drugs.
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